Malaria is one of the major global public health concerns with 1.24 million deaths worldwide in 2010 (Murray, Rosenfeld et al. 2012). Sterile protection against malaria infection can be induced by multiple exposures to radiation-attenuated sporozoite (RAS) parasite forms in mice (Nussenzweig, Vanderberg et al. 1967) and humans (Clyde, Most et al. 1973), if the RAS remain sufficiently viable to invade hepatocytes (Nussenzweig, Vanderberg et al. 1967). Manufacturing of RAS has several technical hurdles to overcome to allow mass immunization (Seder, Chang et al. 2013), and therefore subunit vaccines have been the primary focus of development in recent decades. RTS,S, based on Pf circumsporozoite protein (PfCSP), the predominant sporozoite surface antigen, is the most advanced subunit candidate, but has shown only 31% efficacy against malaria episodes in Phase III testing (Agnandji, Tsassa et al. 2012), and therefore new subunit vaccine strategies are needed. CSP tolerant transgenic mice are also protected after RAS immunization, implicating additional pre-erythrocytic antigens as targets of sterile immunity (Gruner, Mauduit et al. 2007), (Mauduit, Tewari et al. 2010). We therefore sought to identify novel candidate pre-erythrocytic vaccine antigens (PEVA) that could add to the level of protection achieved with CSP immunogens alone. We assume that PEVA candidates are transcribed during liver stage (LS) development, and have used transcripomic data developed in our lab to identify such candidate antigens. LMIV has assessed the protective efficacy of some of these immunogens by DNA vaccination in rodent models of malaria. Our Primary Objective has been to down-select and prioritize the PEVA antigens that had emerged from our transcriptomic studies of liver stage (LS) parasites, and to transition an antigen or antigen combination superior to circumsporozoite surface protein (CSP) alone for evaluation as a PE vaccine antigen (PEVA) candidate in humans. We have achieved this goal by demonstrating significant superiority of CSP in combination with two of our novel antigens, over that of CSP alone, when delivered as DNA vaccinations in our two rodent models: P. yoelii in Balb/c and P. berghei in C57BL/6. We also have found that candidate PEVA antigens are recognized by CD8+ and CD4+ T cells in protected rodents and naturally exposed humans, and last year showed that protection conferred by several of our novel PEVA antigens in mice is dependent on CD8+ T cells, because protection in these mice is lost or redcued when CD8+ T cells are depleted. During the past year, we have initiated extensive immunization and infection studies in mice using virus vectored and DNA vaccines,targeting the leading 8 antigens that have come out of our discovery program. This approach will give us a better understanding of the potential for our candidate antigens to induce sterile immunity that completely prevents infection. In conjunction with our studies, we are partnering with GenVec, a local biotech that has also identified candidate vaccines through their own antigen discovery effort, to comparatively assess candidates from our two programs. Immunizations are ongoing. Related to this ongoing work, we have made the following observations: 1) We showed that native CSP is N-terminally processed in the mosquito host and undergoes a reversible conformational change to mask some epitopes in the N- and C-terminal domains until the sporozoite interacts with the liver hepatocyte. Our findings show the importance of understanding processing and the biophysical change in conformation, possibly due to a mechanical or molecular signal, and may aid the development of a second-generation CSP vaccine. 2) Chemoprophylaxis Vaccination (CVac) confers long lasting sterile protection against homologous parasite strains in humans, and involves inoculation of infectious sporozoites (SPZ) under drug cover. CVac using the drug chloroquine (CQ) induces highly protective immunity of long duration, but it is unclear how CVac induces protective immunity while frequent natural exposure to malaria parasite inoculation by mosquitoes does not. In animal studies, we confirmed that CQ neither eliminates LS parasites nor delays their development. Further investigations into the mechanism of CQ-induced protection after CVac are required, and may give insights relevant to drug and vaccine development. 3) We have previously shown that the HIV protease inhibitor lopinavir-ritonavir (LPV-RTV) and the antibiotic trimethoprim sulfamethoxazole (TMP-SMX) inhibit Plasmodium actively dividing liver stages in rodent malarias and in vitro in P. falciparum, but effect against Plasmodium dormant hypnozoite forms remained untested. Using Plasmodium cynomolgi as an established animal model for the study of liver stages of malaria as a surrogate for P. vivax infection, we demonstrated that neither TMP-SMX nor LPV-RTV kills hypnozoite parasite liver stage forms at the doses tested.